Various assays have been developed to recognize target analytes based on biomolecular probes. The biomolecular probes generally include a capture portion, e.g., nucleic acids or peptides that can selectively bind with targeted biomolecules or biological systems such as cells, tissues and organs. The sensitivities of these bioassays are limited by the binding affinity between the probes and target molecules based on various factors, such as kinetic reaction rate, conjugation chemistry, and steric hindrance, especially when the concentrations of target molecules are low. Currently many biomolecular assays rely on target molecule amplification techniques such as polymerase chain reaction (PCR), which add extra cost and complexity, and limit the speed of the assay. Present biomolecular probes generally also include reporters such as fluorophores, metal nanoparticles, fluorophore clusters, fluorophore-conjugated antibodies/proteins, fluorophore encapsulated nanoparticles, quantum dots and enzymes. Among these reporter probes, solid-core nanoparticle probes, such as fluorophore encapsulated nanoparticles, quantum dots, and metal nanoparticles have high surface-volume ratio, tend to precipitate, aggregate, and nonspecifically bind to the surface or to other biomolecules. The nonspecific binding gives false signals and leads to high noise, limiting the sensitivity, specificity, and dynamic range of the biomolecular assay. Nanoparticle probes are also sensitive to their concentration in the solution, the pH value and the ion concentration. On the other hand, the molecule based reporter probes, such as fluorophores, fluorophore clusters, fluorophore conjugated antibodies, or proteins do not provide strong signals for ultra sensitive biomolecular detection. Enzymes are used extensively in ELISA (Enzyme-linked immunosorbent assay) for signal amplification in antibody-antigen detection. However, because of the non-specific binding, non-specific amplification, and high background noises, the assay cannot reach ultra-high sensitivity, and its dynamic range is usually less than three orders of magnitude.
Attempts have been made to overcome these disadvantages in the past, but generally fall short. For example, U.S. Pat. No. 5,175,270 describes dendritic forms of nucleic acids that offer multiple regions of complementarity to target. U.S. Pat. No. 5,635,352 provides a complicated system of capture probes, capture extenders, label probes and label extenders to multiply the effects specifically on detection of nucleic acids. US2006/0286583 describes multiplexed branched chain DNA assays for detecting two or more nucleic acids. PCT publication WO91/08307 describes enhanced capture of target nucleic acids by the use of oligonucleotides covalently attached to polymers; however, the capture polynucleotides are at the ends of branches of the polymer as opposed to linearly aligned. US2008/0038725 illustrates the use of rolling circle amplification to provide binding sites for a multiplicity of labels after target analytes have been captured. It needs long amplification time after the target analytes have been captured. Moreover, none of these documents suggests combining on the same macromolecular backbone, a multiplicity of binding partners or recognition sites with at least two such sites arranged linearly.
The present invention provides reagents and assays that have advantages over known methods of analyte detection, including high specific binding/capture rate, strong reporting signals, low nonspecific binding rate, and high stability.